Project Summary/Abstract Mammalian heparanase, heparan sulfate degrading endoglycosidase, first cloned and characterized in our laboratory, is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. Heparanase also releases angiogenic factors from the extracellular matrix and tumor microenvironment and thereby induces an angiogenic response in vivo. Moreover, the heparanase protein exerts non-enzymatic activities that further promote tumor angiogenesis, growth, survival and dissemination. Enhanced heparanase expression correlates with metastatic potential, tumor vascularity and reduced postoperative survival of cancer patients. These observations, the anti- cancerous effect of heparanase gene silencing and inhibitory molecules, and the unexpected identification of a single functional heparanase, indicate that the enzyme is a promising target for anti-cancer drug development. The proposed research combines basic and clinical aspects of heparanase enzymatic and non-enzymatic functions, emphasizing, among other aspects, the contribution of heparanase expressed by the tumor microenvironment to cancer progression. Aim 1 focuses on the regulation and causal involvement of heparanase in inflammation associated colon carcinoma, radiation resistance of pancreatic carcinoma, and impact of host- vs. tumor- derived heparanase on cancer progression. Aim 2 focuses on non-enzymatic activities of heparanase, emphasizing the contribution and mode of action of the heparanase C-terminus domain and the involvement of heparanase mediated EGF-receptor activation, in tumorigenesis. The main objective of Aim 3 is to crystallize and establish the 3D structure of the latent and active forms of the heparanase protein and its functional domains. Precise structure/function analysis of the heparanase protein will pave the way for rational design of inhibitory molecules directed against its enzymatic and non-enzymatic functions. The proposed research stems from studies performed during the last 5 years of research supported by the NCI (RO1 CA106456) and the development of molecular tools (i.e., highly purified active and inactive species of heparanase and the respective molecular constructs), inhibitory molecules (i.e., chemically modified non-anticoagulant species of heparin, siRNA), antibodies directed against specific domains of the heparanase protein, assay systems (i.e., in vivo imaging of tumor growth, vascularization and promoter activity, highly sensitive heparanase ELISA and enzymatic activity assays, receptor binding and signal transduction, site directed mutagenesis, ChIP analysis), animal models (i.e., orthotopic models of pancreatic, prostate and inflammation-associated colon carcinoma, heparanase transgenic and knock-out mice) and collaborative arrangements (i.e., crystallization and structural analysis) to carry out and accomplish each of the proposed specific aims.